Coumermycin A1, a coumarin antibiotic, has anticancer, antibacterial, antiviral, and antimalarial activities. We aimed to evaluate the anti-thielerial and anti-babesial activity of coumermycin A1 in mice in vivo. Coumermycin A1 efficacy was determined by the transcription of DNA gyrase, a type II DNA topoisomerase using reverse transcriptase-polymerase chain reaction (RT-PCR) transcription. Coumermycin A1 significantly inhibited the development of preliminary parasitemia (1%). Theileria equi and the Babesia species B. bigemina, B. bovis, and B. caballi were observed with IC₅₀ values of 80, 70, 57, and 65 nM, respectively. Their development was remarkably inhibited at observed concentrations of 10, 25, 50, and 100µM for the studied organisms T. equi, and the Babesia species B. caballi, B. bovis and B. bigemina, respectively. In the subsequent viability test, parasite re-growth was suppressed at 100µM for B. bigemina and B. bovis and at 50 µM for B. caballi and T. equi. Coumermycin A1 Treatment of B. bovis cultures with Coumermycin A1 completely suppressed the transcription of the DNA gyrase subunits B and A genes. In BALB/c mice, the development of Babesia microti was inhibited by 70.73% using 5 mg/kg of Coumermycin A1.